The major specific objective of this proposal is the use of in vitro DNA synthesis systems based on membrane-associated folded chromosomes isolated from E. coil to study the process of initiation of a round of chromosome replication. "Terminated folded chromosomes", isolated via sucrose gradient centrifugation from 1) amino acid-starved E. coli DNA plus cells of 2) from E. coli DNA initiation mutants grown at 43 degrees C, would be used. As an alternative to using folded chromosomes as the DNA substrate, in some experiments E. coli "origin" plasmids, after isolation and characterization, would be used. Criteria based on in vivo initiation studies with thermoreversible E. coli DNA mutants and specific inhibitors would be used to demonstrate that in vitro initiation closely resembled initiation in vivo. The systems so developed would be used to study molecular requirements and regulatory phenomena and as an assay system for isolation of initiation factors. In particular, RNA species (origin-RNA) required for initiation would be isolated and characterized, and their function in initiation would be studied using the in vitro systems. The primary cellular target for transformation of a cell into a malignant cell is the cellular DNA. Further, the overall rate of chromosome duplication is controlled primarily by the initiation process. This project would thus study the molecular basis of that part of the DNA replication process, namely, initiation, most relevant to the cancer problem, demonstrating its direct relevance to the health sciences.